Overexpression of N-glycosylation pathway regulators to modulate glycosylation of recombinant proteins

Abstract

Methods of modulating the properties of a cell culture expressing a protein of interest are provided. In various embodiments the methods relate to the overexpression of proteins involved in the N-glycosylation pathway.

What is claimed is: 
     
       1. A method of regulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture process, comprising:
 transfecting a mammalian host cell to overexpress a protein that is involved in an N-glycosylation pathway, wherein the protein involved in the N-glycosylation pathway is selected from the group consisting of N-acetyl-glucosaminyltransferase-1 (encoded by Mgat1); N-acetyl-glucosaminyltransferase-2 (encoded by Mgat2); a UDP-Galactose transporter encoded by Slc35a2; and combinations thereof, wherein the regulated high mannose glycoform content of the recombinant protein is less than or equal to 10%; 
 wherein the mammalian cell culture is perfused using alternating tangential flow (ATF), and 
 wherein the recombinant protein is harvested and purified. 
 
     
     
       2. The method of  claim 1 , wherein perfusion begins on or about day 1 to on or about day 9 of the cell culture. 
     
     
       3. The method of  claim 1 , wherein perfusion begins on or about day 3 to on or about day 7 of the cell culture. 
     
     
       4. The method of  claim 1 , wherein perfusion begins when the cells have reached a production phase. 
     
     
       5. The method of  claim 4 , wherein perfusion is accomplished by alternating tangential flow using an ultrafilter or a microfilter. 
     
     
       6. A method of regulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture process, comprising:
 transfecting a mammalian host cell to overexpress a protein that is involved in an N-glycosylation pathway, wherein the protein involved in the N-glycosylation pathway is selected from the group consisting of N-acetyl-glucosaminyltransferase-1 (encoded by Mgat1); N-acetyl-glucosaminyltransferase-2 (encoded by Mgat2); a UDP-Galactose transporter encoded by Slc35a2; and combinations thereof, wherein the regulated high mannose glycoform content of the recombinant protein is less than or equal to 10%; 
 wherein the mammalian cell culture is maintained by fed batch, and 
 wherein the recombinant protein is harvested and purified. 
 
     
     
       7. The method of  claim 6 , wherein the culture is fed three times during production. 
     
     
       8. The method of  claim 7 , wherein the culture is fed on a day between day two and four, on a day between day 5 and 7, and on a day between day 8 and 10. 
     
     
       9. The method of  claim 6 , wherein the culture is fed four times during production. 
     
     
       10. The method of  claim 9 , wherein the culture is fed on a day between day two and four, on a day between day 5 and 6, on a day between day 7 and 8, and on a day between day 8 and 10 or later. 
     
     
       11. The method of  claim 1  or  6 , wherein the mammalian cell culture is established by inoculating the bioreactor with at least 0.5×10 6  to 3.0×10 6  cells/mL in a serum-free culture media. 
     
     
       12. The method of  claim 1  or  6 , wherein the harvested and purified recombinant protein is formulated in a pharmaceutically acceptable formulation. 
     
     
       13. The method of  claim 1  or  6 , wherein the mammalian host cell is transfected to express two or more proteins involved in the N-glycosylation pathway, wherein the proteins are selected from the group consisting of Mgat1 and Mgat2; Mgat1 and Slc35a2; Mgat2 and Slc35a2; and Mgat1, Mgat2 and Slc35a2. 
     
     
       14. The method of  claim 1  or  6 , wherein the mammalian host cell has been previously transfected to express a recombinant protein and is subsequently transfected to express a protein that is involved in an N-glycosylation pathway. 
     
     
       15. The method of  claim 1  or  6 , wherein the mammalian host cell is first transfected with a protein that is involved in an N-glycosylation pathway and is subsequently transfected to express a recombinant protein. 
     
     
       16. The method of  claim 1  or  6 , wherein the recombinant protein is a protein comprising an antibody Fc region. 
     
     
       17. The method according to  claim 16 , wherein the recombinant protein is selected from the group consisting of a Fc fusion protein, an antibody, an immunoglobulin, and a peptibody. 
     
     
       18. The method of  claim 17 , wherein high mannose glycoform content of the recombinant protein expressed in the cells that have been transfected to overexpress a protein that is involved in N-linked glycosylation is decreased compared to that produced by the mammalian host cell before being transfected to overexpress a protein involved in N-linked glycosylation. 
     
     
       19. The method of  claim 18 , in which the high mannose glycan species is selected from the group consisting of Mannose 5 (Man5); Mannose 6 (Man6); Mannose 7 (Man7); Mannose 8 (including Mannose 8a and 8b (Man8a and 8b); Man8a and 8b, and Mannose 9 (Man9); and combinations thereof. 
     
     
       20. The method of  claim 19 , in which the high mannose glycoform content of the recombinant protein is less than or equal to 5%. 
     
     
       21. The method of  claim 1  or  6 , wherein the mammalian host cell is transfected to express Mgat1 and Mgat2.